Table 1.

Method for separation and ESI-MS conditions for Pfs25.

HPLC Method	Parameter
HPLC/MS System	Waters Alliance 2695 Waters Q-ToF Premier Mass
Mobile Phase A	0.1% TFAa in water
Mobile Phase B	0.1% TFAa in ACNb
Flow Rate	0.2 mL/min
Column	Waters XBridge BEH 300 C18, 2.1 × 250 mm, 5 μm
Column Temperature	37 °C
Auto sampler Temperature	5 °C
Injector Volume	100 μL
Detector Wavelength	214 nm
Run Time	120 min

	Time (Min)	% Mobile Phase A	% Mobile Phase B
Gradient Program	0	98	2
	3	98	2
	90	60	40
	110	10	90
	112	10	90
	113	98	2
	120	98	2

ESI-MS Method	Parameter
Ionization	Electrospray Positive V Mode
M/Z Scan Range	100-3000 Da
Scan Time	3-112 min
Switch Valve	0-3 min and 112–120 min to waste
	3-112 min to mass spectrometer
Capillary Voltage	3.0 kV
Sample Cone Voltage	30.0 V
Source Temperature	120 °C
Desolvation Temp.	250 °C
MS Condition	MSE
Low Collision Energy	15 eV
Elevated Collision Energy	Ramping from 20 to 90 eV
Scan Time	1.5 s

^aTrifluoroacetic acid.

^bAcetonitrile.